Lactiplantibacillus plantarum P9 for chronic diarrhea in young adults: a large double-blind, randomized, placebo-controlled trial

Chronic diarrhea, affecting 3-20% globally, is characterized by more than three loose stools daily for at least four weeks. Current treatments, such as loperamide and codeine, offer limited efficacy and present side effects like constipation and nausea. The disruption of gut microbial diversity in individuals with diarrhea increases susceptibility to pathogens. Probiotics, live microorganisms conferring health benefits, offer a potential alternative. While some studies have shown improvements in diarrhea symptoms with probiotics, results remain controversial, highlighting the need for further research into specific strains, dosages, and intervention durations. *Lactiplantibacillus plantarum* P9 (P9), a strain isolated from traditional acidic gruel, exhibits properties such as high tolerance to gastrointestinal fluids and potent immunomodulatory activities. This study investigates whether P9 possesses anti-diarrheal properties and its impact on gut microbiota and metabolome.

Existing literature on probiotic treatment for chronic diarrhea presents mixed results. Some studies using single or multi-strain probiotics demonstrated improvements in symptoms like defecation frequency and stool consistency, while others reported no significant improvement in gastrointestinal or psychological symptoms. This variability underscores the importance of considering factors like the specific probiotic strain, dosage, sample size, and study duration. Studies focusing on the gut microbiome in diarrhea have highlighted its disruption and the potential for probiotics to restore balance. The role of the gut phageome, impacting the microbiota and influencing gastrointestinal diseases, is also gaining attention. The multifaceted effects of probiotics, beyond their direct action on symptoms, make investigating their potential for restoring gut balance highly relevant. Previous research on *L. plantarum* strains has demonstrated their effectiveness in alleviating constipation and modulating gut microbial composition and metabolites.

This large, randomized, double-blind, placebo-controlled clinical trial (ChiCTR2000038410) enrolled 189 young adults with chronic diarrhea (defined by Rome IV criteria), randomly assigned to either P9 (n=93) or placebo (n=96). The intervention lasted 28 days, followed by a 14-day follow-up. The primary endpoint was diarrhea symptom severity score (Gastrointestinal Symptom Rating Scale). Secondary endpoints included stool consistency, frequency, urgency, and changes in gut microbiota and metabolome. Fecal samples were collected at baseline, day 28, and day 42 for metagenomic sequencing, untargeted and targeted metabolomics. Data analysis involved Wilcoxon rank-sum tests, covariance analysis, and correlation analysis. The study aimed to analyze the impact of P9 on various aspects of gut health, exploring both clinical symptoms and underlying biological mechanisms.

Both intention-to-treat (ITT, n=189) and per-protocol (PP, n=170) analyses showed a statistically significant reduction in diarrhea symptom severity in the P9 group compared to placebo (20.0%, *P* = 0.050; 21.4%, *P* = 0.048, respectively). PP analysis also revealed a significant reduction in stool consistency score (*P* = 0.050). Metagenomic sequencing revealed significant differences in gut microbiota structure at days 28 and 42 between groups, with P9 increasing levels of beneficial bacteria (*Lactiplantibacillus plantarum*, *Ruminococcus A faecicola*, *Paraprevotella xylaniphila*) and decreasing potentially harmful ones (*Mediterraneibacter torques*, *Coprococcus* sp., *Enterocloster* sp000431375). While phageome alpha diversity showed no significant differences, beta diversity analysis at day 28 showed changes in the phageome composition. Untargeted metabolomics identified 21 differentially abundant metabolites at days 28 and 42, including increased levels of beneficial metabolites like cholate, chenodeoxycholate, and decreased levels of deoxycholate in the P9 group. Targeted metabolomics confirmed increased acetic acid and butyric acid and decreased deoxycholic acid in the P9 group at day 28. The study also found increased CAZymes in the P9 group indicating enhanced carbohydrate metabolism.

This study demonstrates the modest but significant efficacy of *L. plantarum* P9 in alleviating chronic diarrhea symptoms in young adults. The observed improvements are linked to modulations in the gut microbiota and metabolome. The increase in beneficial bacteria and metabolites like SCFAs likely contributed to improved intestinal barrier function and reduced inflammation. The changes in the phageome composition suggest a complex interplay between bacteria and phages in the context of P9's effects. The reduction in deoxycholic acid levels is particularly noteworthy, given its role in diarrhea. The findings support the exploration of targeted probiotic interventions for chronic diarrhea management. The study suggests potential mechanisms linking P9 administration to diarrhea relief, namely through modulating specific gut bacteria, bacteriophages, and microbial metabolites.

This large-scale, randomized controlled trial provides evidence that *L. plantarum* P9 modestly but significantly alleviates chronic diarrhea symptoms in young adults. The multi-omics analyses suggest that this effect is mediated by alterations in the gut microbiota, phageome, and metabolome. While further research with a longer intervention period and inclusion of additional diagnostic tests are warranted, this study supports the potential of P9 as a safe and effective probiotic for managing chronic diarrhea. Future research could focus on longer intervention periods and explore the specific mechanisms underlying the observed changes in the gut microbiome and metabolome.

Limitations include the relatively short intervention period (28 days), the absence of blood parameter assessments to monitor inflammation and gut barrier function, and the lack of detailed dietary information. The study also used Rome IV criteria for chronic diarrhea diagnosis, but lacked further diagnostic tests such as colonoscopy or other laboratory tests, leading to a potential inclusion of individuals with different diarrhea subtypes under the umbrella of ‘chronic diarrhea’. The observed improvements in both placebo and intervention groups suggest a potential placebo effect, although the magnitude of change was greater in the P9 group. The high variability in individual responses to the intervention might have affected the overall findings.